Introduction. This protocol aims to diagnose under laboratory conditions the main Mycosphaerella spp. pathogens of bananas and plantains. The three pathogens Mycosphaerella fijiensis (anamorph Paracercospora fijiensis), M. musicola (anamorph Pseudocercospora musae) and M. eumusae (anamorph Pseudocercospora eumusae) are, respectively, the causal agents of Black Leaf Streak disease, Sigatoka disease and Eumusae Leaf Spot disease. The principle, key advantages, starting plant material and time required for the method are presented. Materials and methods. The laboratory materials required and details of the thirteen steps of the protocols (tissue clearing and in situ microscopic observations, isolation on artificial medium and cloning of single-spore isolate, in vitro sporulation and microscopic observations of conidia, and long-term storage of isolates) are described. Results. Diagnosis is based on the observations of anamorphs (conidiophores and conidia) which can be observed directly from banana leaves or after sporulation of cultivated isolates if sporulating lesions are not present on banana samples.